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identifiers that constitute the sequence divergence at that site, and the height of each letter represents the degree of conservation. Location of motifs (E.coli Phr) is shown below. B.

Schematic representation of motifs in E.coli photolyase. C. Structural mapping of Class I motifs on E.coli Phr crystal structure. Motifs are mapped by colors indicated in A, FAD shown in yellow. D. Enlargement of active site cavity. All three motifs extend into FAD binding site. E. Highly conserved motifs in Class III photolyases. Three out of four motifs are similar to Class I motifs.

0 1 2 3 4 bits

R

N

D

LFYVI R V M L

P

NSG A D EKA V QI R S E V A N PD I E C HSVA G V H

TS

T H E E.coli Phr 226-235 0 1 2 3 4 bits RQ

G

EDSA G

A

WV T Q PS A Q LED A K

R

AT FC

W

G R

W

E.coli Phr 48-54

bases) (Park et al., 1995). Therefore, the three motifs identified by evolutionary trace have proven roles in dimer recognition, chromophore binding, and repair, demonstrating that this approach defines functionally important residues.

Class III signature sequences are similar to Class I CPD photolyase motifs

Reports of photoreactivation in organisms that possess Class III genes exclusively suggest that these proteins possess repair activity. This activity is likely directed towards cyclobutane pyrimidine dimers, since CPDs are the predominant photoproduct formed after UV and therefore contribute most to the reversion in UV-induced killing by photoreactivation (Sancar, 2003). ET analysis of the novel Class III sequences revealed that their most highly conserved sequences resemble Class I photolyases in many aspects (Figure 2.2E). First, Class III proteins possess only a few, highly conserved motifs. Second, the majority of Class III motifs are nearly identical to those found in Class I photolyases. Lastly, all residues that are important in repair of CPDs by Class I photolyases are conserved in Class III genes, providing further support for the hypothesis that these proteins function as CPD photolyases. The sole Class III-specific motif (G⋅X⋅A⋅X⋅R⋅W⋅W) is predicted to map on the

surface of the rear face of the protein, with unknown functional significance.

Characterization of a Class III protein

In order to investigate the function of Class III proteins, a representative sequence from the bacterium C.crescentus was cloned. C.crescentus photoreactivates (Bender, 1984) and possesses only the single Class III gene, therefore making it the candidate photolyase.

C.crescentus Class III Phr was overexpressed and purified as an MBP-fusion protein (Figure 2.3A) in order to test its repair activity in vitro. However, spectroscopic analysis of the purified protein (Figure 2.3 B-C) demonstrated that while the protein retained the MTHF chromophore, it lost the catalytic FAD chromophore during purification. Loss of chromophore binding during purification is common with this family of proteins (Sancar, 2003).

175 83 62 47.5 32.5 25 kDa unind ind MBP-CcPhr

A

B

C

UV Dose (J) Survival rate 1.00E+00 1.00E-01 1.00E-02 1.00E-03 1.00E-04 0 2 4 6 8 10 12 Absorbance 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 300 350 400 450 500 550 600 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 300 350 400 450 500 550 600 Absorbance Wavelength (nm) Wavelength (nm)

D

Absorbance

Figure 2.3 Characterization of C.crescentus Class III photolyase

A. Purification of MBP-CcPhr. Overexpressed protein was purified by affinity chroma- tography on amylose resin. Lane 1, uninduced cells; lane 2, induced cells; lanes 3-4,

increasing amounts of purified MBP-CcPhr. B. Absorbance spectrum of MBP-CcPhr. Peak at 380 nm is indicative of MTHF and possibly FADH°. C. Absorption spectrum of chromophores released from MBP-CcPhr by heat denaturation. Protein was heat-denatured at neutral pH and removed by centrifugation. Upon release from the protein, MTHF is converted to 10-formyltetrahydrofolate, which no longer absorbs at λ > 300 nm; spectrum arises from flavin absorbance, with a predicted peak at 440 nm. D.In vivo photoreactivation by

MBP-CcPhr. Photolyase-deficient E.coli UNC523 cells were complemented with MBP-CcPhr (blue) or empty vector (black) and irradiated with 254 nm UV light at different doses. Cells were either photoreactivated with 365 nm light (dashed lines) or maintained in darkness (solid lines) to assess UV killing. Data are representative of three independent experiments (± SEM).

Unfortunately, photolyase is not active in the absence of FAD, thereby preventing an in vitro

assessment of Class III function using the C.crescentus protein at this time.

To test class III function, we assayed for photoreactivation in vivo by complementation of a photolyase-deficient E.coli strain, an approach that has worked well to characterize other photolyases (Kihara et al., 2004). The UNC523 E.coli strain was transformed with pMalC2-CcPhr or empty vector alone and irradiated with increasing doses of 254 nm UV light to induce photoproduct formation. Cells were either treated with photoreactivating light (365 nm) or kept in darkness to assess total killing by UV, and relative survival was assessed by colony formation assay. Under these conditions, MBP- CcPhr significantly photoreactivated, increasing survival by 15-fold over E.coli transformed with vector alone (Figure 2.3D). Therefore, in combination with previously published in vivo

data from C.crescentus, we can state with confidence that Class III proteins are CPD photolyases.

Signature sequences of DASH cryptochromes resemble those of CPD photolyases

A new class of cryptochromes was identified several years ago, comprised of bacterial genes as well as genes in plants, fungus and aquatic vertebrates. The class, named DASH because its bacterial members were more similar to cryptochromes found in eukaryotes (Drosophila, Arabidopsis, Sy n e c h o c y s t i s and Human) than bacterial photolyases, was suggested to be the progenitor of eukaryotic cryptochromes (Daiyasu et al., 2004). Our lab and another showed that members of this class did not contribute significantly to photoreactivation in vivo, satisfying the definition of a cryptochrome (Hitomi et al., 2000; Worthington et al., 2003); however, one recent study showed that they possess intrinsic, albeit low, CPD repair activity in vitro (Daiyasu et al., 2004). Analysis of the DASH class by evolutionary trace indicates, surprisingly, that DASH cryptochromes are highly similar to CPD photolyases in two aspects (Figure 2.4). First, two of four motifs exhibited significant homology to Class I signature sequences (Figure 2.4B), and map to the FAD

A

B

In document Manual de Buenas Prácticas de Gestión de Servicios para Establecimientos de Hospedaje (página 30-34)